Effect of oxygen supply on metabolism of immobilized and suspended Escherichia coli

The effect of reduced oxygen supply on the production of a recombinant protein (plasmid-encoded beta-galactosidase) was investigated in Escherichia coli. A novel modified bubble tank reactor was used to provide a direct comparison between immobilized and suspended cells in identical environments except for the immobilization matrix. Decreased oxygen supply led to increased beta-galactosidase synthesis by both immobilized and suspended cells. Immobilized cells produced similar amounts of beta-galactosidase as the suspended cells. Lactose consumption and acetate production, on a per cell basis, were significantly higher in immobilized cells, suggesting that immobilized cells utilized fermentative metabolism. However, a transport analysis of the immobilized cell system showed that immobilized cells were not subject to either external or internal mass transfer gradients.     

Effects of caffeic acid phenethyl ester (CAPE) on angiogenesis,apoptosis and oxidatıve stress ın various cancer cell lines

ABSTRACT

Cancer is a common cause of death worldwide. Approximately 80% of cancer patients use complementary or alternative medicines for treatment. Caffeic acid phenethyl ester (CAPE), the main active component of propolis, exhibits cytotoxic, antiproliferative and anti-cancer effects. Despite its anticancer effects CAPE exhibits no known harmful effects toward normal cells. We investigated the effects of CAPE on angiogenesis, apoptosis and oxidative stress using MDA MB-231, N2a and COLO 320 cell lines and CAPE treatments at 24 and 48 h. A two dimensional cell culture system was used and the findings were evaluated by an indirect immunohistochemical method and H-scores were calculated. CAPE was effective for all three cancer cell lines. After 24 and 48 h, we found a significant decrease in live cells and increased stress in the cells based on e-NOS and i-NOS levels.     

Progress in the development of a recombinant vaccine for human hookworm disease: the Human Hookworm Vaccine Initiative

Hookworm infection is one of the most important parasitic infections of humans, possibly outranked only by malaria as a cause of misery and suffering. An estimated 1.2 billion people are infected with hookworm in areas of rural poverty in the tropics and subtropics. Epidemiological data collected in China, Southeast Asia and Brazil indicate that, unlike other soil-transmitted helminth infections, the highest hookworm burdens typically occur in adult populations, including the elderly. Emerging data on the host cellular immune responses of chronically infected populations suggest that hookworms induce a state of host anergy and immune hyporesponsiveness. These features account for the high rates of hookworm reinfection following treatment with anthelminthic drugs and therefore, the failure of anthelminthics to control hookworm. Despite the inability of the human host to develop naturally acquired immune responses to hookworm, there is evidence for the feasibility of developing a vaccine based on the successes of immunising laboratory animals with either attenuated larval vaccines or antigens extracted from the alimentary canal of adult blood-feeding stages. The major antigens associated with each of these larval and adult hookworm vaccines have been cloned and expressed in prokaryotic and eukaryotic systems. However, only eukaryotic expression systems (e.g., yeast, baculovirus, and insect cells) produce recombinant proteins that immunologically resemble the corresponding native antigens. A challenge for vaccinologists is to formulate selected eukaryotic antigens with appropriate adjuvants in order to elicit high antibody titres. In some cases, antigen-specific IgE responses are required to mediate protection. Another challenge will be to produce anti-hookworm vaccine antigens at high yield low cost suitable for immunising large impoverished populations living in the developing nations of the tropics.     

Cell bank characterization and fermentation optimization for production of recombinant heavy chain C-terminal fragment of botulinum neurotoxin serotype E (rBoNTE(H(c)): antigen E) by Pichia pastoris

A process was developed for production of a candidate vaccine antigen, recombinant C-terminal heavy chain fragment of the botulinum neurotoxin serotype E, rBoNTE(H(c)) in Pichia pastoris. P. pastoris strain GS115 was transformed with the rBoNTE(H(c)) gene inserted into pHILD4 Escherichia coli-P. pastoris shuttle plasmid. The clone was characterized for genetic stability, copy number, and BoNTE(H(c)) sequence. Expression of rBoNTE(H(c)) from the Mut(+) HIS4 clone was confirmed in the shake-flask, prior to developing a fed-batch fermentation process at 5 and 19 L scale. The fermentation process consists of a glycerol growth phase in batch and fed-batch mode using a defined medium followed by a glycerol/methanol transition phase for adaptation to growth on methanol and a methanol induction phase resulting in the production of rBoNTE(H(c)). Specific growth rate, ratio of growth to induction phase, and time of induction were critical for optimal rBoNTE(H(c)) production and minimal proteolytic degradation. A computer-controlled exponential growth model was used for process automation and off-gas analysis was used for process monitoring. The optimized process had an induction time of 9 h on methanol and produced up to 3 mg of rBoNTE(H(c)) per gram wet cell mass as determined by HPLC and Western blot analysis.